Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Adeno-associated virus (AAV) is a small (20 nm), replication-defective, non-enveloped virus. Many distinct AAV serotypes have been characterized in human and nonhuman primates. The AAV genome is comprised of single-stranded DNA with 145 bp inverted terminal repeats (ITRs) at both ends. There are two open reading frames (ORFs), rep and cap. While the rep products are essential for AAV replication, 3 capsid proteins (VP1, VP2, and VP3) are expressed from the cap gene. VP1, VP2 and VP3 come together at 1:1:10 ratio to form an icosahedral capsid (Xie Q et al, 2002). During recombinant AAV (rAAV) vector production, an expression cassette flanked by ITRs is packaged into AAV capsid. The genes required for replication of AAV are not included in the cassette. Recombinant AAV is considered the safest and one of the most widely used viral vectors for in vivo gene transfer. The vectors can infect cells from multiple tissue types providing strong and persistent transgene expression. They are also non-pathogenic and have a low immunogenicity profile (High K A, 2011).
One of the immediate goals for gene therapy trials is optimizing vectors to maximize tissue transduction while minimizing the vector dose. Upon entry into the cell, AAV capsid proteins are subject to proteasome mediated degradation. Phosphorylation of surface-exposed tyrosine residues of AAV capsid represents one of the first steps that leads to degradation of the virus via the ubiquitin-proteasome pathway (Zhong L et al, 2007). Most of the regulated proteolysis in the cell occurs through this pathway. Ubiquitin is a small protein (˜8.5 kDa) that can be found in all eukaryotic cells. Ubiquitin is attached to the side-chain of amino-acids of a substrate protein. After additional ubiquitin proteins are attached to the substrate via the initially attached ubiquitin, a polyubiquitin chain is formed and the substrate is marked for degradation (Thrower J S et al 2000, Peng J et al 2003, Bedford L et al 2011). It has been shown that mutation of surface-exposed tyrosine residues leads to an increase in transduction efficiency of AAV2 vectors (Zhong L et al, 2008). More recently, several groups have shown that the strategy is effective also with other AAV serotypes in several tissues, including AAV serotype 6 and 8.
Clearly, a need exists in the art for compositions and methods which improve the transduction of AAV carrying clinically important transgenes in patients in need thereof.